Journal Article

Nephrin Is Expressed on the Surface of Insulin Vesicles and Facilitates Glucose-Stimulated Insulin Release

Jongmin Jeon, Lorenzo Cobianchi, Slavena A. Mandic, Peter Mundel, Luca Inverardi, Camillo Ricordi, Dontscho Kerjaschki, Jochen Reiser, George McNamara, Antonello Pileggi, Karl Tryggvason, Javier Varona Santos, Christina Bark, Alessia Fornoni, Alexandra Jauregui, Kevin Johnson, Per-Olof Berggren, R. Damaris Molano

2010

Overview

Nephrin Is Expressed on the Surface of Insulin Vesicles and Facilitates Glucose-Stimulated Insulin Release Alessia Fornoni 1 , 2 , Jongmin Jeon 1 , Javier Varona Santos 1 , Lorenzo Cobianchi 1 , 3 , Alexandra Jauregui 1 , 2 , Luca Inverardi 1 , Slavena A. Mandic 4 , Christina Bark 4 , Kevin Johnson 1 , George McNamara 1 , Antonello Pileggi 1 , R. Damaris Molano 1 , Jochen Reiser 2 , Karl Tryggvason 5 , Dontscho Kerjaschki 6 , Per-Olof Berggren 1 , 4 , Peter Mundel 2 , 7 and Camillo Ricordi 1 1 Diabetes Research Institute, University of Miami L. Miller School of Medicine, Miami, Florida; 2 Division of Nephrology and Hypertension–Miami Institute of Renal Medicine, University of Miami L. Miller School of Medicine, Miami, Florida; 3 Istituto di Ricovero e Cura a Carattere Scientifico Fondazione Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy; 4 The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden; 5 Cell Matrix Biology, Karolinska Institutet, Stockholm, Sweden; 6 Clinical Institute of Pathology, Medical University, Vienna, Austria; 7 Division of Molecular Medicine, University of Miami L. Miller School of Medicine, Miami, Florida. Corresponding author: Alessia Fornoni, afornoni{at}med.miami.edu . Abstract OBJECTIVE Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic β-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in β-cell function. RESEARCH DESIGN AND METHODS Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic ( n = 5) and nondiabetic ( n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin–transfected cells was used to study nephrin endocytosis. RESULTS Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin–transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release. CONCLUSIONS Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes. Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received May 1, 2009. Accepted October 1, 2009. © 2010 American Diabetes Association

• Publisher: American Diabetes Association
• Subject: Animals / Biological and medical sciences / Cell Membrane - physiology / Diabetes / Diabetes Mellitus - genetics / Diabetes Mellitus, Experimental - genetics / Diabetes Mellitus, Experimental - immunology / Diabetes. Impaired glucose tolerance / Diabetic nephropathies / Diabetic nephropathy / DNA Primers / Endocrine pancreas. Apud cells (diseases) / Endocrinopathies / Etiopathogenesis. Screening. Investigations. Target tissue resistance / Gene Expression Regulation / Glucose / Glucose - pharmacology / Health aspects / Humans / Immunoglobulins / Insulin / Insulin - metabolism / Insulin Secretion / Islets of Langerhans - physiology / Islets of Langerhans Transplantation / Medical sciences / Membrane Proteins - genetics / Mice / Muscle proteins / Original / Pancreas / Pancreatic beta cells / Plasma / Polymerase Chain Reaction / Proteins / Reference Values / Research design / RNA, Small Interfering - genetics / Transfection / Transplantation, Heterologous